Kalanchoe blossfeldiana genomic nanopore
Genomic DNA was extracted using an adapted version of (Keb-Llanes et al., 2002) followed by a further clean up using the ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen, USA). Samples were flash frozen in liquid nitrogen, then 300 mg of leaf tissue was ground in liquid nitrogen, then lysed and homogenised in a mix of two extraction buffers mixed just prior to extraction. 300 µl buffer A (2% Hexadecyltrimethylammonium bromide (CTAB) (w/v), 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 4% polyvinylpyrrolidone (PVP-40) (w/v), 0.1% ascorbic acid (w/v), and 10 mM -mercaptoethanol); and 900 µl buffer B (100 mM Tris-HCl (pH 8), 50 mM EDTA, 100 mM NaCl) were mixed along with 100 µl 20% SDS and 10 mM -mercaptoethanol. Extracts were vortexed then incubated at for ten minutes at 65C while shaking at 1500 rpm. Exactly 410 µl potassium acetate was added, samples were then spun down at 15,300 g for 15 minutes at 4C. Supernatant (1 ml) was transferred to a fresh microcentrifuge tube and added to 540 µl ice cold isopropanol and incubated for 20 minutes. Samples were then washed three times as follows: samples centrifuged at 9600 g for ten minutes at 4C; supernatant discarded, and pellet washed with 540 l 70% ethanol and immediately air dried; pellet was then resuspended in 600 µl TE buffer (10 mM Tris, 1 mM EDTA (pH 8)), 60 µl 3M sodium acetate (pH 5.2) and 360 µl ice cold isopropanol; sample was then incubated for 20 minutes, and the process repeated. After three washes the sample was spun down at 9600 g for ten minutes at 4C then resuspended in 50 µl resuspension buffer (R4, from the ChargeSwitch kit) containing RNase A. The ChargeSwitch protocol followed from this point on, as described in its manual. Short read whole genome library preparation and sequencing of K. blossfeldiana was carried out by Novogene (Beijing, China). Sample and library integrity was tested with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Pair end 150 bp libraries were produced using the NEB Next Ultra DNA Library Prep Kit and sequencing was performed in three runs on the same library on an Illumina NovaSeq 6000.Long read sequencing was carried out over three r9.4.1 minion flow cell using either the RAD004 or RBK004 kits, with libraries prepared as per their protocols. Reads basecalled with Guppy 6.2.1 and subsequently merged.